Znt8 assays for drug development and pharmaceutical compositions

ABSTRACT

The present invention describes methods of identifying drugs for the treatment or prevention of diabetes by measuring the activity of the human zinc transporter ZnT8 and pharmaceutical compositions.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/095,394, filed Oct. 22, 2018, which is a 35 U.S.C. § 371 U.S.national entry of International Application PCT/US2017/029250, having aninternational filing date of Apr. 25, 2017, which claims the benefit ofU.S. Provisional Application No. 62/326,871, filed Apr. 25, 2016, thecontent of each of the aforementioned applications is hereinincorporated by reference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no. R01GM065137-13 from the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The escalating type-2 diabetes (T2D) epidemic is a major global healthconcern. Given that T2D is partly genetically determined, geneticfactors that increase T2D susceptibility enhance related medicalcomplications and lower the life spans of patients with T2D. Recentadvances in genome-wide association studies (GWASs) revived the initialoptimism of identifying T2D susceptibility genes and accelerated thediscovery of these susceptibility gene for diabetes. Genes affectingrisk for T2D have been published including the zinc transporter, member8 (SLC30A8) gene also known at ZnT8. ZnT8 is one or nine human genes ofmulti-spanning transmembrane proteins facilitating Zn²⁺ efflux from thecell and sequestration into intracellular compartments. Within thepancreas, specifically in the islet, there are specific zinc transporter(ZnT8) that mediates zinc enrichment in insulin secretory granules ofpancreatic beta cells. There are known variants of ZnT8 DNA and proteinsequences including a nonsynonymous variant of human ZnT8 (R325W) thatis thought to contribute to the susceptibility of type-2 diabetes (T2D),but it remains unclear how the risk allele correlates with zinctransport.

SUMMARY OF THE INVENTION

This application includes a method of identifying a drug to treat orprevent diabetes in subjects comprising the steps of: providing aproteoliposome with a zinc transporter ZnT8, or functional part thereof;administering an agent to the proteoliposome forming a treated sample;measuring the activity of the zinc transporter ZnT8 in the treatedsample to obtain a first activity measurement and comparing the firstactivity measurement to a second activity measurement of a reference;and identifying the drug when the first activity measurement is lowerthan the second activity measurement. The preferred reference areproteoliposome with a ZnT8 transporter or variant thereof substantiallyfree of agent. A suitable activity of zinc transporter ZnT8 measured inthe present invention is the rate of zinc transport efficiency whereinthe first measurement is from 1.3, 1.4, 1.5, 1.6, 2.0, 2.2, 2.4, 2.8,3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6 to 5.0, 5.2, 5.4, 5.6, 5.8,6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 9.0, 10.0, 11.0,15.0 fold lower than the second measurement and wherein the rate of zinctransport efficiency is based on a Vmax/Km value using stopped-flowkinetics.

Another embodiment of the present invention is a method of identifying adrug to treat or prevent diabetes in subjects comprising the steps of:providing a first sample that expresses a zinc transporter ZnT8, orfunctional part thereof administering an agent to the first sampleforming a treated sample; measuring the activity of the zinc transporterZnT8 in the treated sample to obtain a first activity measurement andcomparing the first activity measurement to a second activitymeasurement of a reference; and identifying the drug when the firstactivity measurement is lower than the second activity measurement.Suitable zinc transporters used in the present invention include thenonsynonymous variant of human ZnT8 (R325W) and a ZnT8 protein orpeptide that has activity and is modified such as with a tag, such as aHis-tag. The sample may be in vitro human cells such as HEK293 cellsthat stably express ZnT8 or a biological sample obtained from a subjectsuch as a biopsy, cells, or tissue. There are alternative ways in whichthe activity of the zinc transporter ZnT8 is measured includingmeasuring the intracellular zinc accumulation in the sample or the rateof zinc transport efficacy wherein the first activity measurement isfrom 1.3, 1.4, 1.5, 1.6, 2.0, 2.2, 2.4, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8,4.0, 4.2, 4.4, 4.6 to 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8,7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 9.0, 10.0, 11.0, 15.0 fold lower than thesecond activity measurement. Typically, the rate of zinc transportefficiency is based on a Vmax/Km value using stopped-flow kinetics. Theagent is selected from the group comprising an antibody, portion of anantibody, nucleic acid, peptide, protein, chemical or combinationthereof.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising a drug of the present invention, salt, solvate,or stereoisomer thereof. The present invention identified lipids thatinhibit the activity of ZnT8, specifically lysophosphatidylcholines,cholesterols, or combinations thereof and example of a drug of thepresent invention.

Another embodiment of the present invention is a method of inhibitingZnT8 activity in pancreas cells of a subject comprising administering tothe subject an effective amount of the drug of the present invention,salt, solvate, or stereoisomer thereof Δn additional step to this methodmay be having the ZnT8 activity measured in a sample taken from thesubject before and after the subject is administered the drug bydetermining the rate of zinc transport efficiency in the sample. Therate of zinc transport efficiency after the subject is administered thedrug is from 1.3, 1.4, 1.5, 1.6, 2.0, 2.2, 2.4, 2.8, 3.0, 3.2, 3.4, 3.6,3.8, 4.0, 4.2, 4.4, 4.6 to 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6,6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 9.0, 10.0, 11.0, 15.0 fold lower thanbefore the subject was administered the drug. The rate of zinc transportefficiency is preferably based on a Vmax/Km value using stopped-flowkinetics.

Another embodiment of the present invention is a method of treating orpreventing diabetes in a subject comprising administering to the subjectan effective amount of the drug of the present invention, salt, solvate,or stereoisomer thereof.

Another embodiment of the present invention is a method of purifying azinc transporter ZnT8 comprising the steps of: providing a crudepreparation of a zinc transporter ZnT8 comprising proteins other thanthe zinc transporter ZnT8; reconstituting the crude preparation of thezinc transporter ZnT8 in a liposome forming a transporter proteoliposomecomprising the ZnT8 in a solution; separating the solution from thetransporter proteoliposone; and forming a purified zinc transporterZnT8. Suitable liposomes used in the present invention comprise a lipidincluding anionic phospholipids, non-bilayer phospholipids, cholesterol,phosphatidylinositol (PI), phosphatidylserine (PS),lysophosphatidylcholine (LPC), cholesterol, or a combination thereof.The crude preparation of zinc transporter ZnT8 is reconstituted in aliposome to maintain the activity of zinc transporter ZnT8 during theseparation step, such as centrifugation, for example. The method of thepresent invention not only creates a highly purified zinc transporterZnT8, but a purified Zinc transporter ZnT8 having a Vmax greater than 4,greater than 5, or greater than 6. The purified zinc transporter ZnT8 ofthe present invention may be in the R-conformation, the W-conformation,or a combination thereof. A crude preparation of zinc transporter ZnT8used in the present invention may be defined as a product of affinitypurification, typically where the affinity purification comprises metalaffinity resins, for example. The methods of the present invention mayinclude additional steps such as detecting anti-ZnT8 autoantibodies in asubject using the purified zinc transporter ZnT8, thereby diagnosingtype-1 diabetes in a subject. The method of the present invention maygenerate anti-ZnT8 mAbs such as humanized anti-ZnT8 mAbs that areconformational specific, for example. Other steps include performingdiagnostic beta-cell imaging using the antibodies of the presentinvention or creating a mAb-based therapeutic ZnT8 inhibitors using theantibodies of the present invention.

The term “activity” refers to the ability of a gene to perform itsfunction such as ZnT8 (a zinc transporter) being able to transport zinc.

The term “express” refers to the ability of a gene to express the geneproduct including for example its corresponding mRNA or protein sequence(s).

The term “reference” refers to a standard or control conditions such asa sample (human cells) or preoteolipisomes with a zinc transporter ZnT8free, or substantially free, of agent.

The term “subject” refers to any individual or patient to which themethod described herein is performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject.

The term “ZnT8” is intended to refer to the (SLC30A8) gene and variantsas well as products including nucleic acid and protein sequences derivedtherefrom. ZnT8 includes modified nucleic acid and amino acid sequencesincluding tags for visualization for example. Examples of ZnT8 nucleicacid and protein sequences suitable for the present invention include:Homo sapiens clone SLC30A8 DNA sequence having a NCBI Accession NumberKR712225.1 and Homo sapiens SLC30A8 protein sequence having a NCBIAccession Number ABQ59023.1 as examples. An example of a ZnT8 genesequence is SLC30A8 solute carrier family 30 member 8 [Homo sapiens(human)] having an NCBI Gene ID: 169026. The ZnT8 used in the presentinvention may be eukaryotic including human and animal or prokaryoticincluding bacterial ZnT8 transporters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D illustrates the induced expression and characterization ofhuman ZnT8 variants. FIG. 1A illustrates Western blotting analysis ofinducible expression of ZnT8 variants. FIG. 1B illustrates intracellularlocalization of ZnT8 expression by immunofluorescence. FIG. 1Cillustrates imaging of intracellular zinc accumulation. FIG. 1Dillustrates the time course of zinc accumulation.

FIG. 2A-2C illustrates the purification of human ZnT8 variants. FIG. 2Aillustrates a homology model of human ZnT8. FIG. 2B illustrates thepurification and reconstitution of ZnT8 variants. FIG. 2C illustratesthe sizing of HPLC profiles of the purified ZnT8.

FIG. 3A-3B illustrates the kinetic analysis of ZnT8 variants. FIG. 3Aillustrates the fluorescence responses to increasing zincconcentrations. FIG. 3B illustrates the steady state kinetics.

FIG. 4 illustrates the functional regulation of ZnT8 by lipids.

FIG. 5 illustrates the time courses of Zimpry-1 fluorescence in responseto the addition of zinc at 0, 5, 19 or 283 nM. The fluorescence responsewere measured in stable expression HEK293 cells with or withoutinduction of the R and W form as indicated. Blk is a negative controlusing parental HEK293 cells with no ZnT8 over-expression.

FIG. 6 illustrates steady state kinetics of the R and W form inreconstituted proteoliposomes with defined compositions as indicated.

DETAILED DESCRIPTION OF THE INVENTION

The islet-specific zinc transporter ZnT8 mediates zinc enrichment ininsulin secretory granules of pancreatic beta cells. A nonsynonymousvariant of human ZnT8 (R325W) contributes to the susceptibility oftype-2 diabetes (T2D), but it remains unclear how the risk allelecorrelates with zinc transport. Here we report a comparative analysis ofa pair of high-risk (R325) and low-risk (W325) variant. The R-form wasfound hyperactive following induced expression in HEK293 cells.Reconstitution of purified R-form into biomimetic membranes yielded a51% increase in the transport rate. During insulin granule biogenesis,the hosting membrane of ZnT8 undergoes enormous lipid remodeling. ZnT8variants were shown functionally tunable to stimulation by anionicphospholipids and inhibition by cholesterol and non-bilayerphospholipids. Over a broad range of permissive lipid compositions, theR-form consistently exhibited accelerated zinc transport kinetics,indicating that the high-risk variant may be targeted for inhibition toreduce T2D risk in the general population.

Zinc forms stable complexes with insulin hexamers, enabling crystallinepacking of the secretory granules of pancreatic beta cells. Defectiveinsulin secretion in the face of insulin resistance is a mainphysiological characteristic of T2D, a complex multifactorialpolygenetic disease with more than 80 T2D-susceptibility loci/genesidentified so far by genome-wide association studies (GWASs). Anonsynonymous single nucleotide polymorphism in SLC30A8 (rs13266634C>T), which causes an arginine to tryptophan change at position 325(R325W), is associated with a 53% increased risk of developing T2D(Sladek R, Rocheleau G, Rung J, Dina C, Shen L, Serre D, Boutin P,Vincent D, Belisle A, Hadjadj S, et al. (2007) Nature. 445, 881-885).The risk allele is widespread in more than 50% of the populationaccording to HapMap data (build 35). SLC30A8 encodes a granular zinctransporter known as ZnT8. In pancreatic beta cells, ZnT8 is highlyexpressed and responsible for transporting cytosolic zinc into insulingranules. However, the molecular mechanism underlying the geneticsusceptibility of ZnT8 polymorphisms remains controversial. ZnT8inactivation in various mouse models revealed large phenotypicvariations ranging from decreased, unchanged to even enhanced insulinsecretion. Over-expression and functional characterization ofpolymorphic alleles in pancreatic beta cells suggested an attenuatedzinc transport activity associated with an increased T2D susceptibility,whereas genotyping rare nonsense and frameshift mutations of ZnT8 inhumans indicated an opposite causal relationship, suggesting that a lossof function of ZnT8 actually reduced T2D risk. The conflicting resultsconcerning the directional relationship between ZnT8 activity and T2Dsusceptibility prevent the identification of specific defects in therisk variant for pharmacological correcting. At present, it is not clearwhether stimulation or inhibition of ZnT8 might be a therapeutic optionto reduce T2D risk in the general population.

Studies of the functional effects of GWAS-identified risk variants inhumans are challenged by small effect sizes that in general do notprovide a clinically useful predictor for disease risk. Althoughprotective effects on T2D risk were observed in loss-of-function ZnT8mutants at an occurrence rate of about 350 carriers out of 150,000genotyped individuals, the extrapolation of the casual relationshipobtained from rare penetrant mutants to common, yet mild ZnT8polymorphic variants is not straightforward, because in humans therelationship between ZnT8 activity and diabetes risk could follow acomplex bell-shaped dose-response. To determine the functional effectsof the common ZnT8 polymorphic variants on T2D risk, we developedinduced expression of human ZnT8 variants in HEK293 cells, purified boththe R- and W-form in a native state, and achieved reconstitution inbiomimetic membranes with defined lipid compositions. A directfunctional comparison of human ZnT8 polymorphic variants at themolecular level sidestepped inherent limitations to phenotypicinterpretation of animal models. Our experiments demonstrated a clearcausality between elevated zinc transport activity and increased T2Drisk.

Induced expression and characterization of human ZnT8 variants. In humanpancreatic islets, ZnT8 expression is confined intracellularly to thebeta cells and alpha cells to a lesser extent. To produce recombinanthuman ZnT8 variants, we generated a series of stable HEK293 cell lines,each with an isogenic integration of a His-tagged ZnT8 variant under atetracycline-inducible promoter. Western blotting using an antibody tothe His-tag showed that the induced R- or W-form reached a similar levelof expression while the uninduced basal level was suppressed below thedetection limit (FIG. 1A). A two-fold increase of post-inductionexpression level was observed for a ZnT8 mutant (termed the AA-form)containing a double Ala-substitution to the predicted transport-site.ZnT8 variants were also monitored for induced expression in HEK293 cellsby immunofluorescence labeling and confocal microscopy. Post-inductionimmunoreactivities toward the His-tag were found predominantlyrestricted to the cytoplasm (FIG. 1B). Additional strong immunostainingof the nuclear envelope was observed due to non-specific antibodybinding as shown by the same staining pattern in uninduced cells and inHEK293 cells with no genomic ZnT8 integration as a negative control.This cell line is referred to hereafter as blank (Blk). The effects ofZnT8 over-expression on intracellular labile zinc concentrations wereexamined using a membrane-permeable zinc-selective fluorescentindicator, Zinpyr-1. Zinpyr-1 labeling produced bright punctate stainingin the juxtanuclear region (FIG. 1C), consistent with the trapping ofZinpyr-1 in the Golgi and acidic subcellular compartments. Cells withinduced expression of the R-form exhibited stronger Zinpyr-1 stainingthan the W- and AA-form. The basal level of Zinpyr-1 fluorescence in Blkwas significant, at a level similar to that of the W- and AA-form (FIG.1C). Quantification of Zinpyr-1 fluorescence in ˜13,000 live cells byflow cytometry yielded a mean fluorescence intensity of 2860.3±9.6 forthe R-form and 2135.4±7.6 for the W-form, respectively. This resultsupported a higher level of vesicular zinc accumulation by inducedexpression of the R-form. We next monitored ZnT8-mediated vesicular zincaccumulation in real time. Stable expression cells or Blk in a 96-wellmicroplate were loaded with Zinpyr-1, and then exposed to a zinc uptakebuffer containing 1 uM pyrithione, a zinc ionophore that was used tobreak down the surface membrane barrier to zinc diffusion, enablingdirect manipulation of the cytosolic free zinc concentration. Zincexposure at 15° C. triggered a linear rise of Zinpyr-1 fluorescencewithin 10 min, which could be quenched by a zinc chelator TPEN. The netfluorescence difference between induced and uninduced cells reflectedZnT8-mediated vesicular zinc accumulation (FIG. 1D). The rate of zincuptake increased progressively with an increasing free zincconcentration from 5 nM to 383 nM. At lower zinc concentrations (5 and19 nM), the rate of three ZnT8 variants showed no significant differenceat a level similar to that of Blk. However, at 383 nM, the R-formexhibited a significantly faster rate of zinc accumulation than theW-form, indicating that the R-form was hyperactive with respect to theW-form. The rates of the W- and AA-form were significantly faster thanthe basal level of Blk, suggesting that both variants were active. At383 nM, the rank order of zinc transport activity was R>W>AA>Blkfollowing induced expression in HEK293 cells (FIG. 1D).Purification and reconstitution of ZnT8 variants. Significant vesicularzinc accumulation observed in Blk (FIG. 5) was attributed to endogenouszinc transport activities of a multitude of zinc efflux and uptaketransporters. The high background fluorescence signal, and a lack of aprecise control over subcellular loading of the zinc indicator limitedthe potential for a quantitative comparison of ZnT8 variants. Todirectly compare zinc transport activities and determine which step(s)in the transport reaction cycle might be affected by the risk allele, weset out to purify ZnT8 variants and to develop functional reconstitutionfor in-depth kinetic analysis. Homology modeling of human ZnT8 based onthe crystal structure of the bacterial zinc transporter YiiP suggestedthat ZnT8 forms a unique Y-shaped homodimer in which two transmembranedomains (TMDs) splay out in the membrane (FIG. 2A). Lipid molecules areexpected to play an essential stabilizing role by filling the void spacebetween two TMDs. Accordingly, we minimized ZnT8 delipidation in thepurification process by coupling affinity purification to liposomereconstitution. The proteins eluted from metal affinity resins containeda major ZnT8 species as shown by SDS-PAGE (FIG. 2B). Most proteincontaminants were not reconstituted, thus could be separated fromreconstituted ZnT8 in proteoliposomes by ultracentrifugation.Re-solubilization of proteoliposomes yielded a mostly pure proteinspecies with slightly reduced mobility on SDS-PAGE (FIG. 2B). Themolecular identities of protein bands before and after reconstitutionwere confirmed by western blotting using antibodies to a N-terminalepitope in ZnT8 and the C-terminal His-tag, respectively (FIG. 2B).

The purified ZnT8 variants were further assessed for protein folding bysize-exclusion HPLC analysis. Despite having a predicted molarabsorptivity of 41535 M⁻¹cm⁻¹, the purified ZnT8 was nearly UV silent,probably due to the high lipid content in the detergent micelles. Todetect ZnT8 elution, we labeled the purified sample with athiol-specific fluorescent probe, fluorescein-5-maleimide, and thenmonitored labeled-ZnT8 by fluorescent and UV detection in tandem.Size-exclusion HPLC analysis of DDM-solubilized proteoliposomes revealeda single fluorescent peak. SDS-PAGE analysis of the peak fraction withcoomassie stain confirmed the presence of purified ZnT8 (FIG. 2B). HPLCre-run of the peak fraction yielded single, mono-dispersed peaks by bothfluorescent and UV detections (FIG. 2C). The apparent molecular weightof the purified ZnT8 was estimated to be ˜120 kDa, in agreement with adimeric assembly of two 36.5 KDa monomers with bound lipids anddetergents. Compared with GFP-tagged ZnT8 in detergent crude extract,the purified ZnT8 without a GFP-tag was slightly right-shifted as aresult of protein size difference (FIG. 2C). The peak profile, however,remained essentially unchanged between the purified ZnT8 and unpurifiedZnT8-GFP, indicating that ZnT8 retained a native fold afterpurification, reconstitution and re-solubilization. For clarity, onlythe HPLC chromatogram of the R-form is shown, as the profiles of the W-and AA-form were essentially identical.

Kinetic difference of ZnT8 variants in reconstituted biomimeticmembranes. Zinc transport by ZnT8 is thought to be a two-step process,initiated by zinc binding to a transport-site followed by a proteinconformational change that moves the bound zinc ion across the membranebarrier. This kinetic process was characterized for bacterial zinctransporters in reconstituted proteoliposomes (Chao Y & Fu D (2004) JBiol Chem 279, 12043-12050). However, reconstitution of human ZnT8variants either caused large vesicle leakage when E. coli polar lipidextract was used, or yielded no detectable transport activity whenbovine liver polar lipid extract was used. To achieve low backgroundleakage and high transport activity, we used a mix of syntheticDioleoylphosphatidyl-Choline (DOPC),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and pure soyphosphatidylinositol (PI) at a ratio (45:20:23:12) that mimicked thephospholipid composition of insulin secretory granules of rat pancreaticbeta cells (INS-1 832/13 cells). ZnT8-mediated zinc transport wasmonitored by Fuozin-3, a membrane impermeant fluorescent zinc indicatorencapsulated in proteoliposomes. ZnT8-mediated zinc influx was about5-10 fold higher than the background leakage measured in liposomes thatwere prepared without protein incorporation (FIG. 3A). The R- or W-formresponded to an increasing extravesicular zinc concentration with ahyperbolic increase of the initial rate of Fluozin-3 fluorescence change(FIG. 3A). Least squares fitting of the steady-state kinetics toMichaelis-Menten equation indicated that the rate of zinc transport(Vmax) for the high-risk R-form was 51% faster, accompanied by a smalldecease of Km (FIG. 3B, Table-1). The Vmax/Km value of the R-form, whichis a measure of zinc transport efficacy, was 2.4 fold higher as comparedto that of the W-form.

The site of R-to-W substitution in human ZnT8 is predicted to be in thevicinity of a cytosolic zinc-binding site in CTD (FIG. 2A). This site ismore than 50 angstroms away from the zinc transport-site in TMD.Mutating the cytosolic zinc-binding site of YiiP was found to impair aCTD conformational change, leading to an allosteric down-regulation ofthe zinc transport activity (17). The increased zinc transport activityof the high-risk R-form is consistent with a functional enhancement viaallosteric over-stimulation. By comparison, the double Ala-substitutionto the transport-site in TMD is predicted to impact zinc transportdirectly, in agreement with a 24-fold Km increase and 59-fold Vmax/Kmdecrease as compared to the corresponding kinetic parameters of theR-form. Previous work showed that Ala-substitution of highly conservedzinc binding residues abolished transport activity of a homologous humanZnTS and a bacterial YiiP. Although the AA-form apparently remainedfunctional at a reduced level, the initial rate of zinc transport seemedlinear with a zinc concentration up to 1.5 mM (FIG. 3B), suggesting apossible nonspecific zinc leakage when the specific transport-site wasdisrupted by a targeted mutation. Thus, the Vmax value of the AA-form(0.028±0.008 s⁻¹) defined an upper bound non-specific leakagecontributing to the kinetic measurement of human ZnT8 variants inreconstituted proteoliposomes.

Functional modulation of human ZnT8 variants by lipids. Duringreplenishment of insulin granules following glucose stimulated insulinsecretion, ZnT8 is trafficked with insulin granule biogenesis en routefrom the endoplasmic reticulum through Golgi networks to insulinsecretory granules. The intracellular trafficking process exposes ZnT8to a dynamic lipid composition as the membranes undergo curvatureformation, granule budding and exocytotic fusion. The remodeling ofinsulin granule membranes results in enrichments of anionicphosphatidylinositol (PI), phosphatidylserine (PS), non-bilayerlysophosphatidylcholine (LPC) and cholesterol. We further investigatedthe functional difference between ZnT8 variants under the influence ofthree major classes of lipids in insulin granules: anionicphospholipids, non-bilayer phospholipids and cholesterol. In almost allmammalian cells, PC accounts for 50% of total cellular phospholipids. PEis the second most abundant phospholipid in mammalian membranes,contributing 20-30% of total phospholipid content. We therefore used a2:1 mixture of DOPC and DOPE to form a minimal biomimetic membrane andthen examined the effect of adding a third anionic phospholipid. Theefficiencies of ZnT8 reconstitution for both variants as well asFluozin-3 vesicular encapsulation remained approximately the same forthe following lipid compositions, DOPC:DOPE (2:1), DOPC:DOPE:DOPG(2:1:1), DOPC:DOPE:DOPS (2:1:1) and DOPC:DOPE:soyPI (2:1:1). The zinctransport activities of both variants in the DOPC:DOPE (2:1)proteoliposomes were severely reduced below the Vmax level of theAA-form at 2.8×10⁻² s⁻¹ (FIG. 4). DOPG, DOPS or soyPI increased Vmax by6.1, 3.5 or 2.7 fold for the R-form, and 2.0, 2.3, or 2.3 fold for theW-form (Table-1). All three anionic phospholipids, regardless of theexact chemical nature of the headgroup, activated zinc transport. Ofnote, the Vmax values of the R-form were consistently larger than theW-form under all conditions of anionic lipid stimulation (FIG. 4). Thezinc transport-site in the homolog model is situated in the outerleaflet of the lipid bilayer and partially accessible to interactionswith surrounding lipid headgroups (FIG. 2A). DOPS and soyPI, bothabundantly found in the insulin granule membranes, seemed to facilitatezinc binding as suggested by a modest decrease of Km values (Table 1).DOPG is not a native anionic lipid in insulin granules with slightlyincreased Km values for both the R- and W-form.

TABLE 1 Summary of kinetic parameters R325 W325 ZnT8 Variant Vmax VmaxLipid compositions (0.01s⁻¹) Km (□M) (0.01s⁻¹) Km (□M)DOPC:DOPE:DOPS:soyPI  6.22 ± 0.38  103.1 ± 22.2 4.07 ± 0.19 162.2 ± 24.4(45:20:23:12) DOPC:DOPE  2.00 ± 0.22 205.4 ± 70.0 2.15 ± 0.15 169.2 ±36.8 (2:1) DOPC:DOPE:DOPG  12.10 ± 0.31 252.9 ± 18.8 4.22 ± 0.22 218.4 ±40.0 (2:1:1) DOPC:DOPE:DOPS  6.99 ± 0.28 165.5 ± 21.1 4.97 ± 0.26 159.2± 30.0 (2:1:1) DOPC:DOPE:soyPI  5.39 ± 0.21 131.0 ± 17.7 4.95 ± 0.16125.2 ± 14.3 (2:1:1) DOPC:DOPE:DOPS:LPC  1.69 ± 0.17  33.0 ± 17.0 2.47 ±0.18  52.3 ± 16.4 (1:1:1:1) DOPC:DOPE:DOPG  10.39 ± 0.53 259.7 ± 36.36.39 ± 0.47 219.3 ± 46.8 (1:1:1) DOPC:DOPE:DOPG  6.39 ± 0.16 418.2 ±25.4 4.36 ± 0.22 416.5 ± 44.9 (1:2:1) DOPC:DOPE:DOPS:soyPI + SM  5.84 ±0.37 125.6 ± 25.2 3.73 ± 0.24 104.8 ± 24.3 (45:20:23 + 12 mol %)DOPC:DOPE:DOPG + cholesterol  2.57 ± 0.32 134.0 ± 56.1 2.59 ± 0.24 160.3± 47.3 (2:1:1 + 10 mol %) DOPC:DOPE:DOPS + cholesterol ND ND ND ND(2:1:1 + 10 mol %) DOPC:DOPE:LPC:DOPS + ND ND ND ND cholesterol(1:1:1:1 + 10 mol %)

Next we examined the effects of two non-bilayer phospholipids, namelythe invert conical LPC and conical DOPE. These non-cylindricalphospholipids alone do not form lipid bilayers, but can be stabilized inthe bilayer structure by the presence of 20-50% cylindricalphospholipids (DOPC, DOPS and DOPG). Adding LPC to 25% or increasing theDOPE concentration in reconstitution mixture did not affect theefficacies of ZnT8 insertion and Fuozin-3 encapsulation, but backgroundzinc leakage slightly increased due to increased bilayer deformation.LPC (Lysophosphatidylcholine, lysoPC) in reconstitutedDOPC:DOPE:DOPS:LPC (1:1:1:1) proteoliposomes reduced the Vmax value ofboth ZnT8 variants below the reference level of the AA-form (FIG. 4).Interestingly, LPC also significantly reduced the Km values of both ZnT8variants (Table-1). The apparent tightening of zinc binding seemed toimpede the transmembrane crossing of bound zinc, resulting in a markeddecrease of Vmax for both ZnT8 variants. LPC is highly enriched ininsulin granules, accounting for 20% of total granule lipids. Wheninserted into the lipid bilayer, the cone-shaped LPC introduced positivecurvature, and the ensuing redistribution of the bilayer lateralpressure might in turn inhibited ZnT8. In contrast, DOPE in the lipidbilayer promoted negative curvature, and increasing the concentration ofDOPE in DOPC:DOPE:DOPG proteoliposomes from a ratio of 2:1:1 to 1:1:1and 1:2:1 had modest effects on the W-form, but progressively reducedthe Vmax of the R-form. Our experiments suggested that significantbilayer curvature changes, either in a positive or negative direction,inhibited zinc transport activity to various degrees.

The third highly enriched lipid class in insulin granule is cholesterol.The compact and conical cholesterol molecules can fit into the voidspace between fatty acid chains, increasing packing density and bendingrigidity of the lipid bilayer. A cholesterol composition probably in therange of 40-50 mol % is required for normal insulin secretion. Manyresidential proteins in the insulin granule have high affinity tocholesterol, likely lowing the actual cholesterol level in the granularmembrane. To examine the effect of cholesterol on ZnT8 transportactivity, we added 10 mol % cholesterol to proteoliposomes made withDOPC:DOPE:DOPG (2:1:1), DOPC:DOPE:DOPS (2:1:1) or DOPE:DOPC:LPC:DOPS(1:1:1:1). The incorporation of cholesterol into proteoliposomes did notaffect ZnT8 reconstitution and Fuozin-3 encapsulation, but invariablyreduced the transport activities of both ZnT8 variants below thereference level of the AA-form (Table-1). The inhibitory effect ofcholesterol explained a complete loss of ZnT8 transport activity inbovine liver polar lipid extract that contained 10 mol % cholesterol.

Recent human population genetics has identified rare loss-of-functionZnT8 variants that confer strong protective effects against T2D. Thisdiscovery validated human ZnT8 as an antidiabetic drug target. Theactual target of therapeutic interventions is the common R325 riskallele in the general population. The translation of rareloss-of-function alleles to therapeutic inhibition of the common riskvariant is questioned by the possibility of a bell-shaped relationshipbetween ZnT8 activity and T2D risk. In this work, we developedquantitative analysis of human ZnT8 variants and characterizedregulatory effects of major lipid components found in insulin secretorygranules. Our experiments showed that the transport activity of humanZnT8 is sensitive to functional modulations by three classes of lipidsrich in insulin secretory granules: the anionic lipids activate whereasLPC and cholesterol strongly inhibit both ZnT8 variants. The high-riskR325 variant is consistently more active than the low-risk W325 variantunder all experimental conditions favoring transport competence (FIG.4). The observed hyperactivity of the high-risk R325 variant mirrors acausal relationship of loss-of-function human mutants to lower T2D risk.Our results suggest that ZnT8 activity is linearly correlated with T2Drisk from a mild ZnT8 polymorphic variant to penetrant loss-of-functionmutants. The association of the human high-risk R325 variant with ZnT8hyperactivity suggests that the over-stimulated R325 variant may betargeted for inhibition to reduce T2D risk in the general population.

Kinetic analysis of human ZnT8 variants in various biomimetic membranesof defined lipid compositions provides insights into functional dynamicsof human ZnT8 in pancreatic beta cells. The inhibitory LPC andcholesterol are both non-bilayer lipids. Their actual concentrations inthe insulin granule membrane are highly dynamic, depending on thepartitioning between residential lipid-binding proteins and the granularmembrane. Rapid turnover of the secretory pathway is a basic functionalrequirement for pancreatic beta cells to maintain homeostatic abundanceof insulin granules. During this process, ZnT8 is trafficked withinsulin granule biogenesis, thus encounters drastically different lipidcompositions from ER to matured insulin secretory granules. ZnT8 iscommonly assumed to pump cytosolic zinc into insulin granules against asteep concentration gradient. However, the abundant presence of stronginhibitory lipids in insulin granules argues for a functionaldown-regulation of granular ZnT8. Secretory granules store anexceptionally high level of zinc in 10 to 30 mM range, while the freezinc concentration in the cytoplasm is kept around a homeostatic setpoint below nM. The inactivation of granular ZnT8 may help reducing zincbackflow out of the zinc-enriched granules. The lipid compositions inthe early secretory pathway seem more permissive for ZnT8-mediated zincaccumulation. Proinsulin is synthesized in ER and loaded with zincduring the transit from ER to cis-Golgi networks. Sorting zinc-bound(pro)insulin into secretory granules could be an alternative mechanismof granular zinc enrichment if ZnT8 is indeed inhibited in the latesecretory pathway. Functional studies of ZnT8 in live beta cells stillawait enabling technologies to track spatiotemporal dynamics of lipidcompositions, subcellular ZnT8 localization and zinc concentrations atthe same time.

Embodiments of the disclosure concern methods and/or compositions fortreating and/or preventing diabetes including Type-1 or Type-2 diabetes.In certain embodiments, the level to which a drug inhibits ZnT8 activityor expression may be any level so long as it provides amelioration of atleast one symptom of diabetes. The level of ZnT8 activity may decreaseby at least 2, 3, 4, 5, 10, 25, 50, 100, 1000, or more fold expressioncompared to the level of a reference, in at least some cases. Anindividual may monitor ZnT8 expression/activity using standard methodsin the art, such as northern assays or quantitative PCR, for example.

An individual known to have diabetes, suspected of having diabetes, orat risk for having diabetes may be provided an effective amount of aninhibitor of ZnT8 activity and/or expression, including lipids. Those atrisk for diabetes may be those individuals having one or more geneticfactors, may be of advancing age, and/or may have a family history, forexample.

In particular embodiments of the disclosure, an individual is given anagent for diabetes therapy in addition to the one or more inhibitors ofZnT8. Such additional therapy may include pharmaceutical agents that arecommercially available, for example. When combination therapy isemployed with one or more inhibitors of ZnT8, the additional therapy maybe given prior to, at the same time as, and/or subsequent to the inducerof ZnT8.

Certain methods of the disclosure provide for methods of diagnosingdiabetes prior to the therapeutic methods of the disclosure, and suchdiagnosis may occur by any methods or means, including at least geneticmarker assay.

Pharmaceutical Preparations. Pharmaceutical compositions of the presentinvention comprise an effective amount of one or more inhibitors of ZnT8such as lysophospatidylcholine, dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcomprises at least one inhibitor of expression and/or activity of ZnT8or additional active ingredient will be known to those of skill in theart in light of the present disclosure, as exemplified by Remington: TheScience and Practice of Pharmacy, 21^(st) Ed. Lippincott Williams andWilkins, 2005, incorporated herein by reference. Moreover, for animal(e.g., human) administration, it will be understood that preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The inhibitor of the activity and/or expression of ZnT8 may comprisedifferent types of carriers depending on whether it is to beadministered in solid, liquid or aerosol form, and whether it need to besterile for such routes of administration as injection. The presentcompositions can be administered intravenously, intradermally,transdermally, intrathecally, intraarterially, intraperitoneally,intranasally, intravaginally, intrarectally, topically, intramuscularly,subcutaneously, mucosally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art.

The inhibitor of activity and/or expression of ZnT8 (includinglysophoshatidylcholine) may be provided to the individual in needthereof by dietary ingesting one or more comestibles that comprise theinhibitor, such as herbs, berries, and/or fruits.

The inhibitor of the activity and or expression of ZnT8 may beformulated into a composition in a free base, neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts,e.g., those formed with the free amino groups of a proteinaceouscomposition, or which are formed with inorganic acids such as forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present disclosure, the composition ofthe present invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a composition contained therein, its use inadministrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with cells of the pancreas, or othermammalian cells such as human cells, for example. The mixing can becarried out in any convenient manner such as grinding. Stabilizingagents can be also added in the mixing process in order to protect thecomposition from loss of therapeutic activity, i.e., denaturation in thestomach. Examples of stabilizers for use in an the composition includebuffers, amino acids such as glycine and lysine, carbohydrates such asdextrose, mannose, galactose, fructose, lactose, sucrose, maltose,sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include inhibition ofactivity and/or expression of ZnT8, one or more lipids, and an aqueoussolvent. As used herein, the term “lipid” will be defined to include anyof a broad range of substances that is characteristically insoluble inwater and extractable with an organic solvent. This broad class ofcompounds are well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure.Examples include compounds which contain long-chain aliphatichydrocarbons and their derivatives. A lipid may be naturally occurringor synthetic (i.e., designed or produced by man). However, a lipid isusually a biological substance. Biological lipids are well known in theart, and include for example, neutral fats, phospholipids,phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids,glycolipids, sulphatides, lipids with ether and ester-linked fatty acidsand polymerizable lipids, and combinations thereof. Of course, compoundsother than those specifically described herein that are understood byone of skill in the art as lipids are also encompassed by thecompositions and methods of the present invention.

One of ordinary skilled in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the inhibitor of activity or expression of ZnT8may be dispersed in a solution containing a lipid, dissolved with alipid, emulsified with a lipid, mixed with a lipid, combined with alipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

Alimentary Compositions and Formulations. In one embodiment of thepresent disclosure, the inhibitors of activity and/or expression ofZnT8, or variants thereof, are formulated to be administered via analimentary route. Alimentary routes include all possible routes ofadministration in which the composition is in direct contact with thealimentary tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered orally, buccally, rectally, orsublingually. As such, these compositions may be formulated with aninert diluent or with an assimilable edible carrier, or they may beenclosed in hard- or soft-shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present disclosure mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

Parenteral Compositions and Formulations. In further embodiments,inducer of expression of ZnT8 may be administered via a parenteralroute. As used herein, the term “parenteral” includes routes that bypassthe alimentary tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered for example, but not limited tointravenously, intradermally, intramuscularly, intraarterially,intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos.6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363(each specifically incorporated herein by reference in its entirety).Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

Miscellaneous Pharmaceutical Compositions and Formulations. In otherpreferred embodiments of the invention, the active compound inhibitor ofactivity and/or expression of ZnT8. or variants thereof, may beformulated for administration via various miscellaneous routes, forexample, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-soluble based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

EXAMPLES/METHODS

Expression constructs. The human ZnT8 isoform-2 cDNA (NM_001172814.1)housed in a pCMV6-entry vector (OriGene Technologies) was shuttled intoa mammalian expression vector pCMV6-AC-GFP with a modified turboGFP tagappended to the C-terminus. The resulting (pZnT8-GFP) was transientlyexpressed in HEK293 cells by Lipofectamine transfection according tomanufacturer's instructions (Thermo Fisher). The human ZnT8 cDNA insertwas further subcloned into a pcDNA5/FRT/TO expression vector that used atetracycline-inducible, hybrid human cytomegalovirus (CMV)/TetO₂promoter to control ZnT8 expression (Life technologies). A hexahistidinetag was added to the C-terminus to facilitate affinity purification. Theresulting His-tagged ZnT8 expression construct (the R-form), pZnT8-His,was also mutated to generated the W-form (R325W mutation) and AA-from(H220A, D224A double mutation) using Q5 Site-Directed Mutagenesis (NewEngland Biolabs). All constructs were confirmed by double strand DNAsequencing.Stable cell lines. Expression cell lines with stable integration ofvarious ZnT8 variants were generated by co-transfection of pZnT8-His andpOG44 at 1:9 ratio into an Flp-In-T-Rex-HEK293 host cell line (LifeTechnologies). This cell line constitutively expressed Tet repressor andcontained a single integrated Flp Recombination Target (FRT) site. Theexpression of an Flp recombinase encoded in pOG44 mediated genomicintegration of the pZnT8-His construct via homologous recombination atspecific FRT sites. The integration also activated the expression of ahygromycin resistant gene, allowing antibiotic selection of stableexpression cell lines. Since all of the hygromycin resistant foci wereisogenic, polyclonal stable cell lines were pooled and used for allexperiments as described below. A separate cell line (Blk) with stableintegration of the empty pcDNA5/FRT/TO vector was generated in parallelas a negative control for immunofluorescence imaging and vesicular zincuptake experiments.ZnT8 expression. Flp-In, T-REx-HEK293 cells stably expressing His-taggedZnT8 variants were grown in Dulbecco's modified Eagle's mediumsupplemented with 10% tetracycline-reduced fetal bovine serum and 100g/ml hygromycin B in 95% air, 5% carbon dioxides at 37° C. Early-passagecells (n<10) with a >95% viability were counted (typically at 1×10⁷cells/ml), seeded at an appropriate density (see below) suited fordifferent experiments on poly-Lys coated surfaces. When cells were grownexponentially to approximately 70% confluent in monolayers, doxycyclinewas added to 1 μg/ml to induce ZnT8 expression. Experiments wereperformed 18-24 hours after expression induction.Immunofluorescence. For visualization of His-tagged ZnT8 expression,stable expression cells were grown on coverslips with or withoutdoxycycline induction. Cells at 50% confluence were fixed with 4%paraformaldehyde, treated with a primary mouse monoclonal antibodyrecognizing the C-terminal His-tag (Abcam, cat. #ab5000, dilution1:100), followed by a goat-anti-mouse secondary antibody conjugated withAlexa Fluor-594 (Thermo Fisher, cat. #A11005, dilution 1:200). Nucleiwere counterstained with DAPI. For visualization of vesicular zincaccumulation, cells were grown on coverslips in glass bottom microwelldishes. Induced cells at 50% confluence were labeled by 5 uM Zinpyr-1added to the culture medium. 30 min after incubation at 37° C., cellswere washed free of excess Zinpyr-1 with Dulbecco's PBS, and thenexposed to 100 uM zinc added extracellularly. Cells were then imagedusing a Zeiss LSM 700 inverted confocal microscope with a 63× oilobjective. Alexa Fluor-594, DAPI and Zinpyr-1 fluorescence were excitedby three separate laser lines (561, 405 and 488 nm), and monitored atrespective emission wavelength ranges under the control of Zen software.Flow cytometry. Stable expression cells were grown in 6-well cellculture plates, induced at 70% confluence, and then labeled with 5 uMZinpry-1 as described above. The labeled cells were washed free ofexcess Zinpry-1 with Hanks' Balanced Salt Solution (HBSS) with glucose,exposed to 100 uM extracellular zinc at room temperature for 30 min.Then, cells were trypsinized, resuspended in ice-chilled HBSS at adensity of 1×10⁶ cells/ml, and kept on ice until running through a flowcytometer. Flow cytometric analyses of vesicular zinc accumulation wereperformed on a MoFlo XDP cell sorter (Beckman Coulter) equipped with a488 nm laser. Data were collected on the forward scatter, side scatter,and 525 nm fluorescence channel. More than 99% of scattering eventsbelonged to a singlet cell population with 98% cell viability based onpropidium iodide staining analysis. Zinpyr-1 fluorescence measured from˜13,000 live cells were used to calculate the mean level of vesicularzinc accumulation for each ZnT8 variant.Vesicular zinc uptake. An uptake buffer with a free zinc concentrationranging from 4.4 to 283 nM was prepared by adding 0.3-1.7 mM ZnSO₄, 1 mMADA, 1 mM EGTA and 1 uM pyrithione to an assay buffer (100 mM NaCl, 20mM HEPES, I mM TECP, pH 7.0). The free zinc concentration in theADA-EGTA dual buffering system was calculated using maxchelator(maxchelator.stanford.edu). Induced cells were loaded with Zinpry-1,trypsinized, resuspended in an ice-chilled plain uptake buffer with noZnSO₄ added. Cells with the expression of each ZnT8 variant or the emptyvector were counted, adjusted to a density of 4×10⁶ cells/ml, anddispensed in 50-□l aliquots to a clear-bottom 96-well microplate(Greiner bio-one). Initial Zinpry-1 fluorescence (F₀) was recorded fromthe bottom on a Flexstation-3 microplate reader (Molecular dynamics),and then zinc-uptakes were initiated by adding 50 ul uptake buffers with2× free zinc concentrations to each well. The Zinpry-1 fluorescenceincreases in response to various free zinc concentrations were recordedat 15° C. in the kinetic mode of Flexstation over a time course of 10min and normalized to Zinpyr-1 loading (F₀). The fluorescence responsesreached a linear phase in 2 min after the uptake temperature approachedequilibrium. The rates of zinc uptake were calculated by linearregression of the Zinpyr-1 signals from 2 to 10 min. All measurementswere performed in 12 replicates, and fluorescence readings were obtainedby averaging over 8-10 measurements that gave consistent results. Mostaberrant measurements occurred in the first and last two columns of themicroplate due to pipetting errors.Purification and reconstitution. 24 hour after induction, early-passagecells (passage number <10) at 90% confluence were scraped on ice andcollected in the assay buffer supplemented with complete mini EDTA freeprotease cocktail tablets as specified by the manufacturer (Sigma). Cellsuspension was homogenized by 40 passages through a high-shear fluidprocessor at 120 psi (Microfluidics), and then the membrane fraction waspelleted by ultracentrifugation at 258 k×g for 60 min. The membranepellet was re-suspended in a solubilization buffer (assay buffer plus25% glycerol). DDM was added to solubilize the membrane according to theweight of the membrane pellet at a DDM-to-membrane ratio of 1:5 (wt/wt).The membrane crude extract was cleared off debris by ultracentrifugationat 258 k×g for 15 min. The supernatant was applied to Talon affinityresins (GE Healthcare) and incubated at 8° C. on a rotary shaker for 45min. The resin was then minimally washed by solubilization buffersupplemented with 25 mM imidazole, and then the immobilized ZnT8 variantwas eluted by increasing the imidazole concentration to 250 mM. Themolecular identity of the eluted ZnT8 variant was validated by westernblotting using two antibodies to the C-terminal His-tag (Cell signalingTechnology, cat. #2365S, catalog number, 1:1000 dilution) and to alinear peptide epitope (Proteintech, cat. #16169-I-AP, 1:500 dilution),respectively. The purity of the eluted proteins was assessed by SDS-PAGEwith imperial protein staining (Thermo Scientific). Preformed liposomeswith a defined lipid composition as indicated were prepared by mixingstock solutions of lipids in chloroform. The lipid mixture was driedunder a stream of nitrogen gas, and traces of chloroform were removed byplacing the lipids under vacuum overnight at room temperature. Driedlipids were rehydrated in a Pyrex borosilicate glass tube with assaybuffer to a lipid concentration of 50 mg/ml, and then sonicated in acup-horn sonicator at 100 W for 2 min (cycles of 10 son, 10 s off) in anice-chilled water bath. The resulting liposome suspension was dilutedwith assay buffer to a final lipid concentration of 7.5 mg/ml with DDMadded at a DDM/lipids ratio of 1:1 (wt/wt). The DDM-liposome mixture wasincubated at room temperature on a rotary shaker for ˜3 hours until agel-like appearance occurred. Reconstitution of ZnT8 variants took placeimmediately after eluted from Talon resins by mixing a ZnT8 variant, oran equal volume of elution buffer, with DDM-destabilized preformedliposomes at an estimated protein-to-lipid wt/wt ratio of 1:200. Thereconstitution mixture was incubated on a rotary shaker at 8° C. for 1hr, then freshly prepared, methanol-washed polystyrene beads (Bio-Beads,SM-2, Bio-Rad) were added to the reconstitution mixture in a 60:1 wt/wtratio to DDM. After incubation overnight at 8° C., the resultingproteoliposomes or liposomes (negative control) were separated from thedetergent-soaked polystyrene beads, and pelleted by ultracentrifugation(258 k×g, 2 hr). The vesicle pellets were resuspended in 0.2 ml assaybuffer with 200 □M Fluozin-3, subjected to 3 freeze-thaw cycles,followed by a 10 second sonication to complete dye encapsulation. Theextravesicular Fluozin-3 was removed by washing vesicles with 3×25 mlassay buffer by three cycles of resuspending and ultracentrifugation(258 k×g, 0.5 hr). Stopped-flow kinetics. Experiments were performed at8° C. on a SFM-3000 stopped-flow apparatus (Bio-logic). Proteoliposomeor liposome samples and an assay buffer containing varyingconcentrations of ZnSO₄ as indicated were loaded into two separatemixing syringes. Zinc influx reactions were initiated by pushing 101 ulfresh reactants at a 1:1 ratio into a mixing chamber at a flow rate of10 ml/s. The reactants were excited at 490 nm, and emissions weremonitored at 525 nm using a 10 nm bandpass cut-off filter. Kinetictraces were recorded over a time course of 10 seconds with instrumentoffset and gain kept constant for all the experiments. All traces werethe cumulative average of 5 successive recordings. Liposome traces werecollected as baselines and subtracted from proteoliposome traces toyield net fluorescence changes ΔF. ΔF/ΔF_(max) was obtained bynormalizing ΔF to the maximum proteoliposome response elicited by anassay buffer containing 3 mM ZnSO₄ plus 2% β-OG. The initial rate ofzinc influx was obtained by linear regression of data points (t<1 s) inthe quasi-linear phase of the initial fluorescence rise. Concentrationdependence data were analyzed by least squares fits of the initialtransport rate to a hyperbola defined by vi=Vamx·M/(M+Km), where Mrepresents the zinc ion concentration, Vmax is the maximum initialtransport rate when the rate of transport approaches to aquasi-stationary state, and Km is the Michaelis-Menten constant. Fits ofexperimental data were preformed using the data analysis softwareSIGMAPLOT (SPSS Inc., Chicago, Ill.).Homology modeling. The protein sequences of the R-allele of human ZnT8isoform-2 and E. coli zinc transporter YiiP were aligned using MODELLER9.16 (https://salilab.org/modeller/). The alignment was imported intoSwiss model (http://swissmodel.expasy.org) to generate a homolog modelof ZnT8 using the crystal structure of YiiP at 2.9-angstrom resolutionas a template (pdb #3H90). The graphic representation of the resultedmodel was prepared using the program PyMol (Delano Scientific).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of identifying a drug to treat diabetes in a subjectcomprising the steps of: (a) providing a proteoliposome comprising azinc transporter ZnT8, or functional part thereof; (b) administering anagent to the proteoliposome forming a treated sample; (c) measuring theactivity of the zinc transporter ZnT8 in the treated sample to obtain afirst activity measurement and comparing the first activity measurementto a second activity measurement of a reference; and (d) identifying thedrug when the first activity measurement is lower than the secondactivity measurement.
 2. The method of claim 1 wherein the reference isthe proteoliposome comprising a ZnT8 transporter or functional partthereof substantially free of the agent.
 3. The method of claim 1wherein the activity of zinc transporter ZnT8 is the rate of zinctransport efficiency.
 4. The method of claim 1 wherein the firstmeasurement is from 1.5 to 5 fold lower than the second measurement. 5.The method of claim 3 wherein the rate of zinc transport efficiency isbased on a Vmax/Km value using stopped-flow kinetics.
 6. A method ofidentifying a drug to treat diabetes in a subject comprising the stepsof: (a) providing a first sample that expresses a zinc transporter ZnT8,or functional part thereof; (b) administering an agent to the firstsample forming a treated sample; (c) measuring the activity of the zinctransporter ZnT8 in the treated sample to obtain a first activitymeasurement and comparing the first activity measurement to a secondactivity measurement of a reference; and (d) identifying the drug whenthe first activity measurement is lower than the second activitymeasurement.
 7. The method of claim 6 wherein the zinc transporter isthe nonsynonymous variant of human ZnT8 (R325W).
 8. (canceled)
 9. Themethod of claim 6 wherein the first sample is in vitro human cells. 10.The method of claim 6 wherein the first sample is a biological sampleobtained from a subject.
 11. The method of claim 6 wherein the activityof the zinc transporter ZnT8 is measured by intracellular zincaccumulation in the sample.
 12. The method of claim 6 wherein theactivity of the zinc transporter ZnT8 is measured by a rate of zinctransport efficacy.
 13. The method of claim 12 wherein the firstactivity measurement is from 1.5 to 5 fold lower than the secondactivity measurement.
 14. The method of claim 13 wherein the rate ofzinc transport efficiency is based on a Vmax/Km value using stopped-flowkinetics.
 15. The method of claim 6 wherein the reference is the firstsample substantially free of agent.
 16. The method of claim 6 whereinthe zinc transporter ZnT8 is a human zinc transporter ZnT8.
 17. Themethod of claim 6 wherein the first sample is HEK293 cells.
 18. Themethod of claim 17 wherein the ZnT8 is a His-tagged ZnT8.
 19. The methodof claim 18 wherein the HEK293 cells stably express the His-tagged ZnT8.20. The method of claim 6, wherein the agent is selected from the groupcomprising an antibody, portion of an antibody, nucleic acid, peptide,protein, chemical, or combination thereof.
 21. The method of claim 20,wherein the agent is a chemical. 22-46. (canceled)